Presently, known methods of creating ions from non-vaporizable molecules fall into two general categories: Electrospray Ionization (ESI) and desorption/ionization from a solid surface.
The ESI technique typically involves spraying a liquid, containing the sample molecules at atmospheric pressure, from a capillary which is at a high voltage relative to an orifice in a sampling plate. A high electric field at the capillary tip from which the liquid flows causes the liquid to become charged. This charged liquid eventually disperses into charged droplets which are drawn towards the sampling plate by the electric field. The region between the capillary tip and the sampling plate is at atmospheric pressure to provide energy to promote desolvation of the droplets. After evaporation of the solvent, either before or after the sampling plate, there are sample ions that may be singly or multiply charged (depending on the structure of the molecule). These sample ions are drawn through the sampling plate orifice into a reduced pressure region of a mass spectrometer due to the flow of background gas from the atmospheric pressure region, between the capillary tip and the sampling plate, to the sub-atmospheric pressure region in the mass spectrometer. Typically, the sub-atmospheric pressure region in the mass spectrometer is at a pressure of less than 10−5 Torr. The sample ions may pass through one or two chambers of intermediate pressure before reaching the high vacuum region of the mass spectrometer.
The most common of the desorption/ionization techniques is Matrix Assisted Laser Desorption Ionization (MALDI) which is most commonly used with a Time-Of-Flight (TOF) mass spectrometer. Typically, the sample and a matrix, such as 2,5-dihydroxybenzoic acid, are both dissolved in appropriate solvents, mixed and deposited on a solid probe surface. The probe effectively becomes an ion source. Once the liquid from the mixture has evaporated, the probe is inserted through vacuum locks into the high vacuum region of a mass spectrometer. A laser beam, often from a 337 nm nitrogen laser, is subsequently pulsed onto the probe surface vaporizing a small amount of dried matrix and sample molecules to form a plume or jet traveling out from the probe surface. The matrix material is specifically chosen to absorb the laser energy in order to rapidly heat and vaporize the sample molecules that it carries. Thus, ionization of at least some of the sample molecules occurs in the plume.
While the detailed mechanisms of vaporization and ionization are not fully understood, most currently accepted models propose that the matrix molecules become ionized in the plume or jet forming a micro-plasma. Neutral sample molecules which are carried away from the probe surface by the expanding micro-plasma then become ionized by charge transfer processes from the matrix ions in the micro-plasma. These processes occur in the micro-plasma only while the matrix ion and sample gas densities are high enough to allow interaction between the matrix ions and the sample molecules. Since the micro-plasma is generated by a laser pulse with a duration of a few nanoseconds focused to an area of less than 1 mm2, the micro-plasma region of each laser pulse is confined to a region very close to the probe surface. In a typical MALDI system, laser pulses are generated at a rate of a few Hz (i.e. 10 or fewer pulses per second). Each generated pulse of ions is accelerated into the TOF mass spectrometer, and a mass spectrum is generated by recording the arrival times of the ions. Usually, the spectra from many laser pulses are added together to create a mass spectrum which can be interpreted.
In the conventional MALDI-TOF configuration described above, the probe surface containing the sample/matrix mixture must be located in a high vacuum region with a typical pressure of 10−6 Torr and more often 10−7 Torr. This is because the TOF mass spectrometer requires high voltages in order to accelerate the ions. Accordingly, a high vacuum is required in order to prevent electrical breakdown in the instrument. In addition, it is very important that the sample ions, formed in the small region close to the probe surface, do not undergo any further collisions with neutral molecules after being accelerated by the high voltage since any further collisions tend to cause the ions to fragment which is undesirable.
A recent development by a group at the University of Manitoba (WO 99/38185) describes the operation of a MALDI ion source in a low vacuum region at a pressure of approximately 10 mTorr (or even up to 1 atmosphere if desired). Ions are generated from a probe surface, as in a conventional MALDI system, but the ions are allowed to collide, at low energies, with a background gas (typically nitrogen) before being introduced into the mass spectrometer. This interaction, often described as collisional cooling, allows the ions to achieve a quasi-thermal equilibrium with the gas which removes all of the original energy of the ions that was induced by the expanding plume from the probe surface. The collisional cooling process also completely decouples the mass spectrometer from the ion source such that ionization parameters such as laser power, the sample's position on the probe surface and the like do not affect the quality of the mass spectrum. The collisional cooling process also converts the pulsed ion stream, formed by the laser pulses of nanosecond duration, into a quasi-continuous ion stream since the ion pulses are stretched in time by collisions with the background gas. After the ions undergo collisional cooling, the ions can be analyzed by any mass spectrometer such as an orthogonal TOF mass spectrometer, a quadrupole or an ion trap.
In any conventional MALDI system that operates at a reduced pressure, the analyte ions must be introduced through a vacuum lock into the source region of a TOF mass spectrometer. However, the MALDI-TOF vacuum locks required for sample introduction add complication and cost. Laiko et al. (U.S. Pat. No. 5,965,884) avoids the problem of vacuum locks by performing the MALDI process at atmospheric pressure. However, this technique suffers ion losses of at least 99% while transferring ions from an atmospheric pressure region to a reduced pressure region.
In the Laiko technique, the surface containing the sample and matrix is located in a region at atmospheric pressure. The surface is also in front of a small orifice that provides a passage to the TOF mass spectrometer chamber. A laser pulse generates ions by the MALDI process at atmospheric pressure and the resulting ion plume is drawn into the TOF mass spectrometer region by a gas flow or an electric field. This technique avoids the necessity of introducing the sample molecules into the vacuum system, however only a small fraction of the sample ions are sampled through the orifice. There are two reasons for the small fraction of ions sampled. The first reason is that the high gas density in the atmospheric pressure region prevents opposite polarity charges, in the micro-plasma of the plume, from separating sufficiently quickly. These opposite charges then recombine which changes a sample ion to a sample molecule thereby reducing the sample ion intensity. The second reason is that the diameter of the orifice that connects the atmospheric pressure region to the vacuum region in the TOF mass spectrometer must be very small so that vacuum pumps can maintain the high vacuum necessary for the operation of the TOF mass spectrometer and pumping requirements are kept reasonable. Accordingly, the resulting poor sampling efficiency through this small orifice reduces the sensitivity of this method compared to the conventional MALDI process discussed above.
Although the details of the MALDI process are not fully understood, most researchers agree that the ionization efficiency is very low, i.e. only 0.1 to 0.01% of the deposited sample molecules are actually converted into ions in the laser-created plasma. It seems likely that many sample molecules are carried away from the probe surface as a neutral species and are simply pumped away by the vacuum pumps. Therefore, if these sample molecules could be ionized, the sensitivity of the method would be greatly increased.
Franzen et al. (U.S. Pat. No. 5,663,561) attempted to address the very low ionization efficiency of the MALDI process by using a laser to desorb the matrix/sample mixture in an atmospheric pressure region and separate, unipolar reagent ions from a corona discharge to subsequently chemically ionize these sample molecules at atmospheric pressure. It is known that conventional atmospheric pressure chemical ionization (APCI) efficiencies can approach nearly 100% (under favorable thermodynamic conditions). In a conventional APCI source, the sample to be ionized is in a gaseous form. The gaseous sample then flows through a region where reagent ions are created. Under conditions where the reagent ions and sample gas are well mixed and where the interaction time is relatively long (i.e. several milliseconds or longer), the ionization efficiency can be very high.
In particular, Franzen teaches that the material from the vaporized MALDI plume is drawn through a corona discharge region. The vaporized matrix/sample ions are mixed with the reagent ions from the corona discharge in a tube connected to a small hole in a sample plate. The resulting ions are then transferred into the vacuum region of the mass spectrometer. However, similarly to the Laiko method, ions must still be transferred through a small orifice into the vacuum chamber for analysis typically by a TOF mass spectrometer. This configuration results in poor sample ion transmission efficiency. As such an ion loss of up to 99% occurs which reduces the practical utility of this method for trace analysis.
Accordingly, it is desirable to provide a method and an apparatus that results in a more sensitive MALDI process that can be used with a mass spectrometer system.